1. Field of the Invention
The present invention relates a method of efficiently producing polyhydroxyalkanoates (hereinafter abbreviated as PHAs) by means of microorganisms. More particularly, the present invention relates to a method of producing PHAs having a desired chemical structure, efficiently as compared with conventional methods, from substituted fatty acid esters as a raw material by using microorganisms capable of producing PHAs having a substituent derived from the raw material in a side chain and accumulating it in their cells.
2. Related Background Art
Many microorganisms have already been reported to produce poly(3-hydroxybutyric acid) (hereinafter abbreviated as PHB) or other PHAs and to accumulate those products in their cells (Biodegradable Plastics Handbook, edited by Biodegradable Plastics Society, published by NTS, pp.178-197). Like conventional plastics, those polymers can be utilized in production of a variety of products through melt processing or the like. Further owing to the biodegradability, those polymers have the advantage that they can completely be decomposed by microorganisms in nature. Consequently, unlike many conventionally used synthesized polymer compounds, they do not remain in the natural environments to cause pollution, nor need to be incinerated, and therefore they are advantageous in terms of prevention of air pollution and global warming. Further, they have an excellent biocompatibility and are highly expected to be used as soft materials for medical use.
It has been well-known that the microorganism-produced PHAs may have a variety of compositions and structures depending on the types of microorganisms used for the production as well as and the compositions of the culture media and the conditions for culturing them, and mainly from a viewpoint of improving the physical properties of PHAs, investigations have been performed regarding how to control the compositions and structures.
For example, it has been reported that Alcaligenes eutrophus H16, ATCC NO. 17699 and its mutant strains produce copolymers of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB) and 3-hydroxyvaleric acid (hereinafter abbreviated as 3HV) in various composition ratios by changing the carbon sources in their culture (Japanese Patent Publication No. 6-15604, Japanese Patent Publication No. 7-14352, Japanese Patent Publication No. 8-19227 and the like).
Further, Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IFO 13852 produces a polyester comprising 3HB and 4-hydroxybutyric acid as monomer units by culturing with gluconic acid and 1,4-butanediol as carbon sources.
Incidentally, PHAs disclosed in these documents are referred to as ‘usual’ PHA because of having no side chain other than methylene or ethylene side chains.
On the other hand, Japanese Patent gazette No. 2642937 discloses that Pseudomonas oleovorans ATCC 29347 produces PHAs comprising monomer units of 3-hydroxyalkanoates (hereinafter abbreviated as 3HAs) of 6 to 12 carbon atoms by supplying acyclic aliphatic hydrocarbons as carbon sources.
Japanese Patent Application Laid-Open No. 5-74492 discloses a method for producing copolymers of 3HB and 3HV by bringing microorganisms, Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. into contact with primary alcohols of 3 to 7 carbons.
Japanese Patent Laid-Open No. 5-93049 and Japanese Patent Laid-Open No. 7-265065 disclose that binary copolymers of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HHx) are produced by culturing Aeromonas caviae in oleic acid or olive oil as carbon sources.
Further, certain types of microorganisms are reported to produce PHAs into which a variety of substituents including, for example, unsaturated hydrocarbons, ester group, allyl group, cyano group, halogenated hydrocarbons, epoxide and the like are introduced, and it has been tried to improve the physical properties of microorganism-produced PHAs by employing such a technique.
For example, Makromol. Chem., 191, 1957-1965, 1990, Macromolecules, 24, 5256-5260, 1991, Chirality, 3, 492-494, 1991, and the like report that Pseudomonas oleovorans produces PHAs containing 3-hydroxy-5-phenylvaleric acid (hereinafter abbreviated as 3HPV) as a monomer unit and some changes in the polymer physical properties are found, which are attributable to the fact that 3HPV is contained.
Incidentally, PHAs having side chains as disclosed in these documents are referred to as ‘unusual’ PHAs in this specification.
Conventionally, microorganism-produced PHAs having a variety of compositions and structures have been obtained by changing the types of microorganisms to be employed for the production, the culture compositions and the culturing conditions. The purposes of the trials are mainly to improve the physical properties of PHAs as plastics by changing the ratios of a plurality of 3HAs contained in the PHAs.
On the other hand, the unusual PHAs into which substituent groups are introduced into their side chains as described above are expected to be developed for wide applications as functional polymers having remarkably useful, functional properties attributed to the characteristics of the introduced substituents. It is therefore supposed to be extremely useful and important to develop innovative polymers provided with such functional properties in addition to the biodegradability, microorganisms capable of producing and internally accumulating such polymers, and efficient production methods of such polymers using such microorganisms.
Incidentally, the methods generally employed for producing such unusual PHAs into which a variety of substituent groups are introduced as side chains, that is, PHAs containing monomer units represented by the following chemical formula (2), by microorganisms, comprise steps of chemically synthesizing a substituted fatty acid having the following chemical formula (6) having the substituent to be introduced, culturing microorganisms in the synthesized fatty acid, and then extracting the produced PHA, as the above reported examples using Pseudomonas oleovorans. (wherein R represents an arbitrarily selected substituent and x represents an integer of 0 to 8.) (wherein R represents an arbitrarily selected substituent and x represents an integer of 0 to 8.)
For example, Polymer Prepr., 35, 627-628, 1994 and Can. J. Microbiol., 41, 32-43, 1995 report that PHAs were produced by the method illustrated in the following reaction scheme. (wherein x represents 2 or 4.)
That is, at first, 6-(4-cyanophenoxy)hexanoic acid ethyl ester is synthesized by Williamson reaction of 4-cyanophenol and 6-bromohexanoic acid ethyl ester. Then, the 6-(4-cyanophenoxy)hexanoic acid ethyl ester is hydrolyzed under an alkaline condition to obtain 6-(4-cyanophenoxy)hexanoic acid. Finally, the obtained 6-(4-cyanophenoxy)hexanoic acid is supplied to Pseudomonas oleovorans, which is one of PHA producing bacteria, to culture the bacterium and the product is extracted to obtain a PHA containing 3-hydroxy-6-(4-cyanophenoxy)hexanoic acid as a monomer unit.
However, in the general production methods of the unusual PHAs involving steps of chemically synthesizing a substituted fatty acid and supplying it to microorganisms as described above, the chemical synthesis of the raw materials, i.e. the substituted fatty acids, requires several steps of chemical reactions to result in considerably complicated steps for the production and in many cases, it takes a long time, costs a great expense and requires troublesome works. Further, depending on the types, the number, and the loci of the substituents, there possibly exists a trouble such that the hydrolytic decomposition reaction of an ester does not smoothly proceed in the synthesizing process of the substituted fatty acid, which has posed various problems in the unusual PHA production in industrial scale and in the apparatus development for the production.